Adhesion of cells to an extracellular matrix is mediated primarily by integrins expressed on the cell surface. Integrins are transmembrane receptors expressed on a wide variety of cells. Ligands for integrins include adhesive extracellular matrix proteins such as fibronectin, vitronectin, collagen and laminin. While purified extracellular matrix components have been used as a coating for the culturing of living cells, in purified form these components do not form the desired three dimensional matrix which characterizes extracellular matrices as they occur in vivo.
A three-dimensional basement membrane-derived extracellular composition obtained from the EHS (Engelbreth Holm-Swarm) tumor grown in mice is disclosed in U.S. Pat. No. 4,829,000. This composition when reconstituted resembles interconnected thin sheets of the lamina densa of the basement membrane to which cells attach. This same basement membrane can be provided by a continuous or immortalized mammalian cell line having the phenotypic properties of EHS murine tumor cells (U.S. Pat. No. 5,354,666) thereby advantageously eliminating the use of mice to maintain the EHS tumor cells. However, the composition is still characterized by significant disadvantages in that it is derived from the EHS tumor, which is of mouse origin. Further, the tumor can not be manipulated to produce specific biologically active growth factors which can be vitally important to the growth, maintenance and differentiation of difficult cells.
Extracellular matrix contains proteins known to support the attachment of a wide variety of cells: fibronectin, vitronectin, thrombospondin, collagens, decorin, laminin and heparin sulfate are examples of matrix proteins. In addition to providing a scaffold for cell attachment, matrix can serve as a reservoir of ligands for growth factor receptors. A number of growth factors including insulin-like growth factor, fibroblast growth factor, members of the transforming growth factor beta family, vascular endothelial growth factor and hepatocyte growth factor have been shown to bind to extracellular matrix proteins and heparan sulfate. For example, Jones et al (J. Cell Biol 121:679-687 (1993)) describe the association of IGFBP-5 with ECM produced by human fetal dermal fibroblasts and in particular its binding to collagen Types III and IV, laminin and fibronectin as detected by polyclonal antisera assays. In addition, these growth factors may be stored in a latent form within the ECM, wherein they become activated and effect cell growth, differentiation or behavior. Still other-investigators (Park et al) have shown that degradation of ECM, produced by human embryonic kidney cells grown on plastic, can release or mediate FGF growth factor activity (Mol. Bio Cell 4:1317-1326 (1993)). It is an aspect of the present invention to provide procedures for controlling the amount and type of growth factors present in ECM and not to rely on serendipitous, uncontrolled production as investigators have traditionally done and in particular to be able to control the amount and presence of FGF, BMP and IGF-1 alone and in co-mixture in ECM.
Growth factors tend to be highly unstable both in vivo and in vitro. This characteristic has hampered studies designed to elucidate their effects on cells cultured in vitro and as biological response modifiers when administered therapeutically to laboratory animals in vivo. In attempts to improve delivery in vivo, synthetic delivery systems have been prepared by incorporating active agents such as growth factors within a vehicle containing matrix material. Previous efforts are exemplified by the use of hydrophobic polymers such as ethylene vinyl acetate (Gospodorwicz et al J Cell Phys 128:475-484, 1984). Hunziker (U.S. Pat. Nos. 5,206,023; 5,270,300; 5,368,858) disclose the use of matrix materials such as fibrinogen, collagen, Sepharose and gelatin. Other investigations have utilized collagen-binding ligand-active agent matrices for improved growth factor delivery in vivo. Vuori et al (U.S. Pat. No. 5,830,504) discloses the formation of a synthetic matrix by conjugating a biodegradable polymer to an alpha V-beta3 ligand and a growth factor receptor ligand. In these conventional approaches, the composition were prepared synthetically by mixing or incorporating the purified active agent into the vehicle with the intent of delivering such agent in vivo.
It is an aspect of the present invention to avoid the unnatural characteristics imposed by such approaches. It is another aspect of the present invention to provide for preparing complex extracellular matrices containing active agents (such as growth factors) for the cultivation of difficult cells in vitro.
While one product, Matrigel .TM. offered by Becton Dickinson and Company (N.J.), purports to provide some of these advantages by providing a plate having deposited thereon ECM with growth factors incorporated, this product contains poorly characterized growth factors in terms of regulation of the type and quantity of growth factors present. As a consequence, this product is more of a research curiosity and is difficult to use in a controlled fashion.
It is yet another further aspect of the present invention to improve upon such products and to provide methods for producing ECM on surfaces having predetermined growth factors or mixtures thereof in known concentrations.
It is still another aspect to provide ECM with desired growth factors incorporated therein which will be more useful for research and production purposes than conventional approaches.
Additional understanding of the state of the art and general knowledge of the skilled artisan may be had by reference to the following, which, like the references cited elsewhere, are fully incorporated herein:
U.S. Patent Documents:
U.S. Pat. No. 4,829,000, May, 1989, Kleinman, et al. PA1 U.S. Pat. No. 5,354,666, October, 1994, Danielson et al. PA1 U.S. Pat. No. 5,770,448June, 1998, Jones, et al
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